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Katharina Müller, Rainer Heintzmann, Jost Hillenkamp, Christine A Curcio, Thomas Ach; Organelle distribution and intracellular autofluorescence (AF) spectra in human retinal pigment epithelium (RPE) cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):30. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
RPE organelles (lipofuscin LF, melanolipofuscin MLF, melanosomes M) accumulate with age and exhibit blue light AF. While the total AF-spectrum of RPE cells has been characterized (PMC4285597), the possibility of spectra specific to different organelle subpopulations suggests that multi-layer spectral analyses of individual cells could deliver further insights into LF/MLF/M physiology. Here, we report serial sectioning spectral data of human RPE cells.
Thirty-five previously prepared RPE flatmounts (PMC4123894; normal: 10 <51 yrs, 10>80 yrs, 15 AMD) were imaged at three eccentricities (fovea, perifovea, near periphery) using two imaging modalities: 1) super-resolution structured illumination microscopy (SIM, Zeiss Elyra.S1); 2) high-resolution confocal laser scanning spectral microscopy (Zeiss LSM780). The same RPE cells were viewed on both devices, acquiring serial sections (488 nm exc. apical to basal, step size: 100 nm in SIM, 390 nm in LSM). The numbers of AF granules (SIM) and spectral emission (LSM; em 490-695 nm; 8.9 nm spectral channel width ) in individual cells were analyzed using FIJI.
Analysis of 3 specimen (2 normal, 1 AMD) shows delineation and distribution of individual LF, MLF, and M in all SIM sections. It explains well the AF intensity profile (Fig 1C, weak-AF M apical and LF and MLF more centrally). Spectral images of the same RPE cells show a broad emission apically that narrows to a spectrum with peaks and shoulders centrally, and then broadening again basally (Fig 1D).
This is the first study imaging spectra in serial sections of individual human RPE cells. In addition, SIM delivers important information on LF/MLF/M distribution. Sectional spectrum changes are indicative of a highly organized organelle accumulation/degradation process. Whether age or AMD impact subcellular spectra is basis of ongoing studies with larger sample sizes.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
SIM (A) and LSM (B) images of the same (1-3) RPE cells (88 y/o donor, unremarkable macula). A/B Images cropped from a z-scan, at the apical side of the cells. SIM clearly displays LF/MLF/M distribution and content. (C) AF-intensity z-profiles. Cells 1 and 3 with fewer M than cell 2 show brighter AF that is more apically located. (D) Spectra of single layers of a z-scan (cell 1). An apical broad spectrum narrows and shows peaks (↑) and shoulders (↓) centrally, then broadens again basally.
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